THE DNA CLEAVAGE PATHWAY OF IRON BLEOMYCIN - STRAND SCISSION PRECEDESDEOXYRIBOSE 3-PHOSPHATE BOND-CLEAVAGE

DNA strand scission initiated by bleomycin is a multistep process. Thr ee C-C or C-O bonds are broken, releasing base propenal, a nucleic bas e derivative with deoxyribose carbons 1-3. Either C-3'-(phosphate-O) c leavage or C-3'-C-4' plus C-1'-(ring-O) bond cleavages could cause str and cleavage. To determine the sequence of bond breakage, d(CAAGCTTG) duplex was examined for rates of 1) strand scission, monitored by the hyperchromicity of cleavage-induced denaturation; 2) base propenal for mation, monitored by H-1 NMR spectroscopy; 3) 5'-terminal phosphomonoe ster formation, monitored by P-31 NMR spectroscopy. Strand scission oc curred with t(1/2) = 4.1 +/- 0.5 min at 4 degrees C, faster than base propenal formation (t(1/2) = 6.7 +/- 0.3 min). Thus newly cleaved DNA includes a base propenal precursor (t(1/2) = 2-3 min). The 5'-phosphat e terminus forms (t(1/2) = 7.4 +/- 0.8 min) concurrently with base pro penal. Since strand scission precedes phosphomonoester formation, stra nd scission cannot arise from C-3'-(phosphate-O) cleavage. Instead, th e base propenal precursor must be linked to the future 5'-phosphate te rminus, with strand scission arising from a combination of C-3'-C-4' a nd C-1'-(ring-O) bond cleavages. These results provide experimental su pport for a recently proposed mechanism that accommodates an early oxy gen attack at C-4' and 2'-deprotonation without requiring simultaneous strand scission and 5'-phosphate terminus formation.